I want to add RNase A to remove RNA from the IP test sample. How do I set the conditions for RNase treatment? At 37 °C for 30 minutes, I am worried that it will affect the protein. At 4 °C, I am worried that RNase will not fully function.
Assuming these are just straight cell/tissue lysates, RNAse may be unnecessary. RNA is horrendously labile, and most RNA isolation protocols take specific efforts to protect RNA against spontaneous degradation even by endogenous RNAses, let alone ones you've added post-hoc.
In other words, while you are preparing your lysates (with protease inhibitors, I hope), your RNA is probably being rapidly degraded anyway.
If you wanted to make absolutely sure, I would just add a dab of RNAse to your lysis buffer and otherwise continue as normal. 30mins at 37 is massive overkill.
RNAse A is active at room temperature. We use a 5 minute incubation at room temperature to remove RNA from our samples.
30 min at 37 degree will not affect protein but I also agree with John that this step is not necessary for remove RNA if you are doing protease inhibitor treatment.
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